Technical variability is greater than biological variability in a microarray experiment but both are outweighed by changes induced by stimulation.

Bryant PA, Smyth GK, Robins-Browne R, Curtis N - PLoS ONE (2011)

Bottom Line:
A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated.Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components).Variability in gene expression was very low and likely to improve further as technical advances are made.

Affiliation: Department of Paediatrics, The University of Melbourne, Melbourne, Australia.

ABSTRACT

Introduction: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated. This study quantified the variability in gene expression at each level of a typical in vitro stimulation experiment using human peripheral blood mononuclear cells (PBMC). The primary objective was to determine the magnitude of biological and technical variability relative to the effect being investigated, namely gene expression changes resulting from stimulation with lipopolysaccharide (LPS).

Methods and results: Human PBMC were stimulated in vitro with LPS, with replication at 5 levels: 5 subjects each on 2 separate days with technical replication of LPS stimulation, amplification and hybridisation. RNA from samples stimulated with LPS and unstimulated samples were hybridised against common reference RNA on oligonucleotide microarrays. There was a closer correlation in gene expression between replicate hybridisations (0.86-0.93) than between different subjects (0.66-0.78). Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components). There was variability in gene expression both at baseline and after stimulation with LPS and proportion of cell subsets in PBMC was likely partly responsible for this. However, gene expression changes after stimulation with LPS were much greater than the variability from any source, either individually or combined.

Conclusions: Variability in gene expression was very low and likely to improve further as technical advances are made. The finding that stimulation with LPS has a markedly greater effect on gene expression than the degree of variability provides confidence that microarray-based studies can be used to detect changes in gene expression of biological interest in infectious diseases.

pone-0019556-g005: Effect of LPS and sex on gene expression with log2 ratios contributed by each component along the x and y axes respectively.

Mentions:
There is almost no overlap between genes that respond to LPS and genes that are expressed differently between the sexes (figure 5). The genes most differentially expressed between the sexes were XIST (represented by 2 spots on the microarray) and DOM3Z (figure 5). XIST is a gene expressed only on the inactivated X chromosome, therefore only in females [10]. DOM3Z is a gene found in the MHC III region paired with RP1, a protein thought to be involved in androgen-responsiveness in male mice [11] which could explain its sex effect. However, the effect of LPS on their expression was minimal. Although not designed to investigate the effects of sex on LPS response, further analysis showed that this effect was not strong in this study.

pone-0019556-g005: Effect of LPS and sex on gene expression with log2 ratios contributed by each component along the x and y axes respectively.

Mentions:
There is almost no overlap between genes that respond to LPS and genes that are expressed differently between the sexes (figure 5). The genes most differentially expressed between the sexes were XIST (represented by 2 spots on the microarray) and DOM3Z (figure 5). XIST is a gene expressed only on the inactivated X chromosome, therefore only in females [10]. DOM3Z is a gene found in the MHC III region paired with RP1, a protein thought to be involved in androgen-responsiveness in male mice [11] which could explain its sex effect. However, the effect of LPS on their expression was minimal. Although not designed to investigate the effects of sex on LPS response, further analysis showed that this effect was not strong in this study.

Bottom Line:
A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated.Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components).Variability in gene expression was very low and likely to improve further as technical advances are made.

Affiliation:
Department of Paediatrics, The University of Melbourne, Melbourne, Australia.

ABSTRACT

Introduction: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated. This study quantified the variability in gene expression at each level of a typical in vitro stimulation experiment using human peripheral blood mononuclear cells (PBMC). The primary objective was to determine the magnitude of biological and technical variability relative to the effect being investigated, namely gene expression changes resulting from stimulation with lipopolysaccharide (LPS).

Methods and results: Human PBMC were stimulated in vitro with LPS, with replication at 5 levels: 5 subjects each on 2 separate days with technical replication of LPS stimulation, amplification and hybridisation. RNA from samples stimulated with LPS and unstimulated samples were hybridised against common reference RNA on oligonucleotide microarrays. There was a closer correlation in gene expression between replicate hybridisations (0.86-0.93) than between different subjects (0.66-0.78). Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components). There was variability in gene expression both at baseline and after stimulation with LPS and proportion of cell subsets in PBMC was likely partly responsible for this. However, gene expression changes after stimulation with LPS were much greater than the variability from any source, either individually or combined.

Conclusions: Variability in gene expression was very low and likely to improve further as technical advances are made. The finding that stimulation with LPS has a markedly greater effect on gene expression than the degree of variability provides confidence that microarray-based studies can be used to detect changes in gene expression of biological interest in infectious diseases.